Mitochondria are the main sources of energy in the cell. They contain their own DNA (mtDNA), whose genes encode components of athe respiratory chain/oxidative phosphorylation system. They are essential for the normal functioning of all cells in the body, and are absolutely critical for the function of those tissues that are highly dependent on aerobic metabolism, especially muscle and brian. Since 1988, both mtDNA point mutations and mtDNA rearrangements (i.e. large-scale deletions [delta-mtDNAs] and duplications [dup-mtDNAs]) have been associated with a heterogeneous group of mitochondrial encephalomyopathies. This Continuing Competitive Application proposes to follow up on our currently-funded efforts on sporadic mtDNA deletions, and to expand our efforts to include mtDNA duplications, which are associated not only with sporadic disorders, but with mendelian- and maternally-inherited disorders as well. We propose to address two related issues. First, we will study the relationship between mtDNA duplications and mtDNA deletions in the pathogenesis of mitochondrial diseases. We will transfer mtDNA duplications (dup-mtDNAs) to mtDNA-less cells (human pomicron cells) and create cytoplasmic hybrid (cybrid) lines harboring different proportions of dup-mtDNAs. These cells will be analyzed for relevant mitochondrial functional characteristics in order to understand athe relationship between phenotype and genotype. We will also try to determine if dup-mtDNAs coexist with delta-mtDNAs in vivo and if dup-mtDNAs can give rise to delta- mtDNAs. Second, we will study genetic complementation of delta-mtDNAs, by fusing two respiratory-deficient cybrids containing two non-overlapping homoplasmic populations of delta-mtDNAs,a nd selecting for cells in which wild-type function has been restored, due presumably to fusion of mitochondria and to complementation between the two genomes. We also propose to create a mouse model of delta-mtDNA diseases, by using mouse cybrid clones harboring a large proportion of a specific species of delta-mtDNA (derived from aged mice, which are known to accumulate low levels of delta-mtDNAs) for injection into fertilized mouse embryos. The proposal to create a "transmitochondrial" mouse model is deemed exploratory in nature. Although this idea is fraught with potential problems, the potential payoff is great, and we would like to include it as an ancillary aim of our work.